The conversion of arachidonic acid to prostaglandins and other eicosanoids is controlled by the two well known cyclooxygenase (COX) isoforms COX-1 and COX-2. COX-2 is an inducible isoenzyme that can be up-regulated in numerous pathologic conditions, including inflammation and cancer. Blocking COX activities with non steroidal anti-inflammatory drugs (NSAIDs) is a widely adopted clinical strategy for the treatment of inflammatory related diseases and pain. Main adverse-effects associated with the chronic usage of classical NSAIDs, are gastrointestinal serious side-effects and renal toxicity. Selective COX-2 inhibitors, though lacking for the great part the gastrointestinal toxicity typical of classical NSAIDs, have recently highlighted undesirable cardiovascular life threatening adverse-effects. An alternative to NSAIDs is the use of corticosteroids, however also in this case chronic use can result in severe side effects.
Since approaches that target gene transcription may complement or even be more successful than the enzyme inhibition, exploration of strategies to specifically block COX gene expression was the object of a remarkable scientific efforts in the last decade (R. G. Ramsay, Int. J. Immunopathol. Pharmacol., 2003, 16 (2S), 59-67). Recently, it was reported that some NSAIDs (including Celecoxib) exert part of their action directly on COX-2 transcriptional regulation, explaining why such agents display greater effects on this isoform than enzyme inhibition data would suggest (K. S. Chun, Biochem. Pharmacol. 2004, 68, 1089). Up-regulation of COX-2 is mediated by a variety of stimuli including cytokines, tumour promoters, oncogenes and growth factors. Intracellular signalling pathways which can induce and regulate COX-2 expressions are complex, and due to cell system dependence are still poorly understood. However, growing evidences suggests that IL-1β and COX-2 play a crucial role in the pathogenesis of inflammatory diseases and tumour growth. In the most of tissues, IL-1β induced COX-2 over-expression is mediated by stimulation of either protein kinase C (PKC) or Ras signal transduction system which enhances mitogen-activated protein kinase (MAPK) activity, which in turn activates transcription of COX-2 along with other cytokines. It has been recently demonstrated, in several tissues, that the three MAP kinases (p38, JNK and ERK 1/2) are involved in controlling COX-2 expression and translation. For instance, this was reported for human chondrocytes stimulated with IL-1β (N. Nieminen Mediators of inflammation, 2005, 5, 249-255), or in human colorectal cancer cells stimulated with IL-1β (W. Liu, Cancer Research, 2003, 63, 3632; Cellular Signalling, 2006, 18, 1262), and in renal mesangial cells (J. Biol. Chem. 1998, 273, 28670).
Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by articular synovitis leading to cartilage degradation and bone erosion. Rheumatoid synovium shows over expression of COX-2 which in turn give rise to massive production of PGE2, responsible for vasodilatation, fluid extravasations and pain. Among a variety of mediators affecting COX-2 expression IL-1β appears to be the main triggering agent (Arthritis Research, 2005, 57).
Osteoarthritis (OA) is the most common form of arthritis, and is largely recognized to be a frequent cause of serious disability in older adults. Synovial inflammation characterized by mononuclear cell infiltration, proliferation of new blood vessels, production of pro-inflammatory cytokines and other mediators of joint damage has been highlighted in the synovial tissues from patients with early and late OA (Ann Rheum. Dis., 2005, 64, 1263-67), and the importance of synovitis in the pathophysiology of OA is increasingly recognized (Haywood, Arthritis Rheum., 2003, 48, 2173; Shibakawa, Osteoarthritis Cartilage, 2003, 11, 133). Increased expression of cytokines, COX-2, adhesion molecules and angiogenic factors are characteristics of chronic synovitis. It has been shown how PGE2 produced by COX-2, in human osteoarthritis explants modulates cartilage proteoglycan degradation, thus highlighting to this inflammatory mediator not only a role in propagating the inflammation process but also a direct involvement in tissue degeneration (Arthritis Rheum., 2002, 46, 1789). It has also been demonstrated how, in OA synoviocytes the mechanism of IL-1β induction for COX-2 expressions follows the same signalling pathway above discussed for the chondrocytes (Arthritis & Rheum., 2004, 50, 2829).
Elevated levels of COX-2 expression have been detected along with high levels of IL-1β, in patients affected by inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, where the inflammatory/autoimmune response is triggered by an exaggerated response to antigens produced by the gut bacteria.
Expression of COX-2 has been reported to be elevated in human colorectal adenocarcinoma and other tumors, including those of breast, cervical, prostate and lung. Genetic knock-out or pharmacological inhibition of both COX-2 and/or its expression has been proven to protect against experimentally-induced carcinogenesis. Accordingly, the inhibition of abnormally or improperly elevated levels of COX-2, by blocking the enzyme and/or its expression, provides also one of the most effective and promising strategies for cancer chemoprevention.
Blockage of cytokines in inflammatory diseases, has lead to the greatest advances in medicine of recent years. Tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) are important biological entities collectively referred to as pro-inflammatory cytokines, which play a role in several diseases, for example such as toxic shock syndrome, RA, OA, diabetes and IBD. In these diseases, chronic elevation of inflammation exacerbates or causes much of the observed pathophysiology. Pro-inflammatory cytokines play a decisive role in the generation of the inflammatory and destructive cartilage degeneration as well as bone erosion in arthritis (B. Moller, Springer Semin. Immunopathol., 2006, 391). Due to resulting structural damage, bone erosion is a major reason for disability in arthritis patients. Bone erosion in arthritis is a consequence of synovial osteoclast formation (B F. Boyce, Curr. Opinion Rheumatol, 2006, 18, 427). Inflammatory cytokines produced in inflamed synovium induce in the bone marrow the release of osteoclast precursors, which reached the inflamed joints and in response to cytokine stimuli differentiate into the bone-resorbing osteoclasts. Thus, pro-inflammatory cytokines in arthritis are responsible for both the progression and diffusion of the inflammatory status within the joint as well as the bone damage. Antagonists of IL-1β have been shown to reduce the degradation of cartilage matrix components in a variety of experimental models of arthritis. Interleukin-6 (IL-6) is a pro-inflammatory cytokine, prevalently expressed in activated monocytes and macrophages, which plays a fundamental role in many chronic inflammatory diseases, particularly implicated in the acute phase response and critically involved in the maintenance of the disease state (J. Scheller, Scand. J. Immunol., 2006, 63, 321). Overexpression of IL-6 has been implicated in the pathology of IBD, arthritis (RA and OA), asthma, colon cancer, multiple myeloma, post-menopausal osteoporosis.
A number of anti-cytokines therapies are currently in clinical trials, and several monoclonal antibodies against TNF and recombinant soluble TNF receptor (Etanercept, Enbrel) as well as recombinant soluble IL-1 receptor (Anakinra, Kineret) reached the market, demonstrating a pronounced activity in treating diseases such as RA, IBD and Crohn's disease. These biological high-molecular weight products, which are based on the antagonism of the circulating cytokine, are however expensive, limited to parenteral administration route and can give rise to immunogenic adverse effects, likely due to their biological nature.
Strategies aimed at blocking cytokine production with small molecules are still of great therapeutic interest, since could be more efficient in blocking cytokine circulation, not endowed with the immunogenic adverse effect of the biological product, less expensive and of simpler administration route. In addition, simultaneous blockade of COX-2 production and pro-inflammatory cytokines production should interrupt the self propagating loop which has been found relevant for the triggering and maintenance of the pathology in inflammatory diseases.
As discussed above, inflammation causes the induction of COX-2, leading to the release of prostanoids, which sensitize peripheral nociceptor terminals and produce localized pain hypersensitivity, however peripheral inflammation also generates central sensitization by direct widespread induction of COX-2 expression in spinal cord and CNS neurons, which results in an increased neuronal excitability and pain hypersensitivity (J. Neurochem., 2003, 86, 318).
Although arthritis (OA and RA) is defined as inflammation of the joints, the primary feature with which patients present in the clinic is chronic pain; even though arthritis is not the only pathology which can give rise to chronic pain, it is rather common and quite representative of this kind of pain. Chronic pain can be divided into inflammatory pain, a kind of pain more related to peripheral tissue damage/inflammation, and neuropathic pain. Neuropathic pain refers clinically to a group of chronic pain syndromes. These syndromes share the common feature that they are caused by an initial nerve damage, which subsequently results in an abnormal sensory processing in the central and peripheral nervous system. Neuropathic pain conditions are the consequence of a number of diseases, for instance diabetes, cancer, amputees, multiple sclerosis.
Peripheral sensitization and central sensitization are the two major mechanisms underlying the generation of pain. When tissue damage occurs, mechanisms in both the nervous and the immune system trigger the release of sensitizing agents such as pro-inflammatory prostaglandins (PGE2), 5-HT, bradykinin, histamine, ATP, cytokines from inflammatory cells and nerve terminals. These mediators evoke activation of specific ion channels through the excitation of peripheral nociceptive neurons, involving activation of intracellular kinases, and resulting in peripheral sensitization. Activation of peripheral nociceptors also reflects in a dependent neuronal plasticity in the CNS. This plasticity modifies the performance of nociceptive pathway by enhancing and prolonging the responses to subsequent peripheral stimuli. These changes in the spinal cord, as well as in the brain are referred to central sensitization. Central sensitization plays a major role in maintaining elevated pain sensitivity and it is responsible for the pain produced after injury by normally innocuous low threshold afferent inputs. A so complex mechanism for pain induction and control can explain why the treatment of pain conditions has not found yet a satisfactory pharmacological solution.
In order to identify effective agents for the clinical management of pain, several alternate pharmacological approaches have been carried out in the last decade, for example COX-2 inhibitors, displayed a good efficacy in the treatment of inflammatory pain, but lacked effectiveness in the treatment of neuropathic pain, in addition for COX-2 inhibitors the undesirable life threatening side-effects mentioned above suggest not to use these drugs for clinical management of chronic pain. The available analgesics for the treatment of neuropathic pain, for instance some tricyclic antidepressant (e.g.: Amitriptyline) and a few antiepileptic drugs (e.g. gabapentin, lamotrigine, and carabamazepine) are effective in some patients, however there is still a large need for efficient drugs for neuropathic pain treatment.
Pharmacological agents acting at controlling cytokines and PGE2 expression can counteract the above described mechanisms of peripheral and central sensitization thus acting as efficient and potent analgesics (M. Schafer, Immune Mechanisms of Pain and Analgesia, pg. 41-50 Plenum Publishers, 2003).